scholarly journals Expansion, isolation and first characterization of bovine Th17 lymphocytes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Patricia Cunha ◽  
Yves Le Vern ◽  
Christophe Gitton ◽  
Pierre Germon ◽  
Gilles Foucras ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Free Cell ◽  
Protein Levels ◽  
Interleukin 17A ◽  
Th17 Lymphocytes ◽  
Ifn Γ ◽  
Bovine Species

Abstract Interleukin 17A-producing T helper cells (Th17) are CD4+ T cells that are crucial to immunity to extracellular bacteria. The roles of these cells in the bovine species are poorly defined, because the characterization of bovine Th17 cells lags behind for want of straightforward cultivation and isolation procedures. We have developed procedures to differentiate, expand, and isolate bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation with antibodies to CD3 and CD28, we expanded IL-17A-positive CD4+ T cells in a serum-free cell culture medium supplemented with TGF-β1, IL-6 and IL-2. Populations of CD4+ T cells producing IL-17A or IFN-γ or both cytokines were obtained. Isolation of IL-17A-secreting CD4+ T cells was performed by labelling surface IL-17A, followed by flow cytometry cell sorting. The sorted Th17 cells were restimulated and could be expanded for several weeks. These cells were further characterized by cytokine profiling at transcriptomic and protein levels. They produced high amounts of IL-17A and IL-17F, and moderate amounts of IL-22 and IFN-γ. The techniques developed will be useful to characterize the phenotypic and functional properties of bovine Th17 cells.

scholarly journals Development and characterization of IL-21–producing CD4+ T cells

2008 ◽  
Vol 205 (6) ◽  
pp. 1369-1379 ◽  
Author(s):  
Akira Suto ◽  
Daisuke Kashiwakuma ◽  
Shin-ichiro Kagami ◽  
Koichi Hirose ◽  
Norihiko Watanabe ◽  
...  
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
The Other ◽  
Intracellular Staining ◽  
Autocrine Growth ◽  
Autocrine Growth Factor ◽  
Ifn Γ

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21–producing CD4+ T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21–producing CD4+ T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21–producing CD4+ T cells without the induction of IL-4, IFN-γ, IL-17A, or IL-17F production. On the other hand, TGF-β inhibited IL-6– and IL-21–induced development of IL-21–producing CD4+ T cells. IL-2 enhanced the development of IL-21–producing CD4+ T cells under Th17-polarizing conditions. Finally, IL-21–producing CD4+ T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21–producing CD4+ T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6–rich environment devoid of TGF-β, and that IL-21 functions as an autocrine growth factor for IL-21–producing CD4+ T cells.

IL-17A-Expressing CD4+ T Cells Must Acquire the Expression of T-Bet and IFN-γto Destroy Tumor Cells In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 468-468
Author(s):  
Pawel Muranski ◽  
Sid P Kerkar ◽  
Zachary A Borman ◽  
Robert Reger ◽  
Luis Sanchez-Perez ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Efficacy ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
End Effector ◽  
Polarized Cells ◽  
Ifn Γ

Abstract Abstract 468 We have recently demonstrated that Th17-polarized TCR transgenic CD4+ T cells specific for TRP-1 melanoma antigen are superior to Th1-polarized cells in mediating effective anti-tumor responses against advanced disease after adoptive transfer. The therapeutic activity of Th17-skewed cells is critically dependent on their ability to secrete IFN-γ, suggesting that the Th17 subset might evolve in vivo. However, the developmental program of Th17-polarized cells in vivo remains substantially un- elucidated. We developed a novel TCR-transduction technique that enabled us to rapidly confer specificity for a cognate antigen upon any population of T cells, regardless of its genetic background, its previous polarization history or its state of differentiation. Using adoptive transfers into tumor-bearing hosts, we were able to study the functionality of these genetically-engineered T cells in vivo. In vitro, CD4+ T cells cultured in type 17 conditions acquired end-effector phenotype (CD62Llow, CD45RBlow), but proliferated slower than cells grown in type 1 condition. Thus, we hypothesized that Th17-polarized cells might represent a less mature, more central-memory like subset. This notion was supported by their ability to secrete high quantities of IL-2 and higher expression of IL-7 receptor. In contrast, Th1-polarized cells upon in vitro re-stimulation upregulated PRDM1 that encodes BLIMP1, a molecule associated with the end-effector senescent phenotype. Moreover, Th1-skewed cells overexpressed caspase 3 and were prone to activation-induced cell death as measured by annexin V assay, while type 17 cells were resistant to apoptosis, and robustly expanded in secondary cultures. Using the TCR gene transfer technique we tested the treatment outcomes when Th17-polarized cells deficient for IL-17A were used. In contrast to wild-type (WT)-derived Th17 cells that effectively eradicated established tumors, we observed significant impairment of treatment with IL-17A-deficent cells. Similarly, we observed reduction in treatment efficacy when CCR6-deficient Th17 cells were transferred. CCR6 is a receptor for CCL20, a chemokine highly induced Th17 cells and thought to contribute to the trafficking of those cells to the site of inflammation. In both cases however, the addition of exogenous vaccination and IL-2 significantly improved treatment efficacy. Thus, we concluded that Th17-associated factors play the role in the anti-cancer activity of type 17 cells. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-skewed cells derived from IFN-γ-deficient CD4+ cells as well as from t-bet-deficient mice, which are not able to develop type 1 responses. In contrast to WT-derived Th17 effectors, IFN-γ-deficient cells did not show any anti-tumor activity, while t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the capacity to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Overall, here we demonstrate that TCR gene engineered Th17-polarized cells can efficiently treat advanced tumor. The high activity of in vitro-generated anti-tumor Th17 cells relies on the contribution of type 17-associated characteristics, including both the secretion of inflammatory factors IL-17A and CCL20, as well as the superior capacity to survive and expand upon the secondary stimulation. Importantly however, type 1-defining t-bet-mediated plasticity in the lineage commitment is required for the full therapeutic effect, underscoring the dualistic nature of Th17-skewed cells. Disclosures: No relevant conflicts of interest to declare.

Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 711-711
Author(s):  
Srimoyee Ghosh ◽  
Sergei B Koralov ◽  
Irena Stevanovic ◽  
Mark S Sundrud ◽  
Yoshiteru Sasaki ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Autoimmune Encephalitis ◽  
Cell Types ◽  
Wild Type ◽  
Ifn Γ

Abstract Abstract 711 Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells. NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases. We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls. To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells. To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease. In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures: No relevant conflicts of interest to declare.

Functional Characterization of CD4+ T-Cells in Aplastic Anemia (AA)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1340-1340 ◽  
Author(s):  
Shahram Y Kordasti ◽  
Judith C. W. Marsh ◽  
Sufyan Al-Khan ◽  
Jie Jiang ◽  
Alexander E Smith ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Group ◽  
High Throughput ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Absolute Number ◽  
Th2 Cells ◽  
Th1 Cells ◽  
Ifn Γ ◽  
Pre Treatment

Abstract Abstract 1340 We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p<0.001). Absolute numbers of Th1 and Th2 cells in all pre-treatment patients were higher compared to HDs (6.4 × 107/L v 1.8 × 107/L)(p=0.03) for Th1 and (2.6 × 107/L v 2.4 × 106/L)(p=0.006) Th2 cells. Although mean percentages of AA Th17 cells were higher than in HDs (1.5% v 0.15%)(p=0.04), differences in absolute numbers were not significant. Absolute numbers of Th2 and Th17 cells were increased in SAA (1.3 × 107/L v 7.4 × 106/L for Th2)(p=0.01) compared to NSAA (5.7 × 106/L v 2.15 × 106/L for Th17)(p=0.02). Ratios of Th1/Tregs (p=0.003), Th2/Tregs (p=0.02), and Th17/Tregs (p=0.001) were higher in SAA and VSAA compared to NSAA. Percentage of both activated (CD4+CD45RA−CD25highFoxp3high) and resting (CD4+CD45RA+ CD25highFoxp3low) Tregs was decreased in AA patients, compared to HDs (p=0.004 and p=0.01), whereas cytokine secreting Tregs (CD4+CD45RA−CD25high Foxp3low) were increased in AA (p<0.003). Sorted Tregs from AA patients did not suppress cytokine secretion by autologous or HD T effectors (Te) cells in 1:1 co-cultures, whereas IL-2 and IFN-γ secretion by AA Te (CD4+CD25lowCD127high) was suppressible by allogeneic Tregs from HDs, confirming Tregs dysfunction. AA Tregs did not inhibit either CD154 or CD69 expression on Te cells. Tregs from AA patients secreted significantly more IFN-γ, TNF-α and IL-17 (p=0.02, p=0.02 and p=0.01, respectively) after 4 hours stimulation with PMA/Ionomycine compared to HDs. Expression levels of FoxP3, ROR□c and T-bet in AA Tregs was normal. IFN-γ secreting cells (Th1) were enriched using enrichment kit then further enriched by FACS sorting. CDR3 region products of TCR Vβ-chain were amplified using Vβ specific forward and Cβ reverse primers. CDR3 PCR products from AA patients and HDs were subjected 454 sequencing (Roche GS FLX titanium). Sequencing was performed to yield an average ‘depth’ in excess of 1000 clonally reads (1000x) for each sample specific CDR3 PCR amp icon. Reads were processed using Roche Amp icon Variant Analyzer software (AVA). Diversity of TCR receptors (measured by spectratyping and confirmed by high throughput deep sequencing) in AA Th1 cells was lower than HDs (p=0.037), as shown by the percentage and number of consensus clusters in total sequence reads. Interestingly, percentages of the most dominant CDR3 clones, revealed by high throughput sequencing, were higher in AA compared to HDs, regardless of spectratyping pattern. Global gene expression of Tregs was compared in 3 pre-IST AA patients and 5 HDs. A unique gene signature consisting of 86 genes that were significant was identified. There were 8 down regulated genes (fold change) in the pre-treatment group; PIN4 (−4.1), OR2T12 (−3.3), AMAC1 (−2.73), PERP (−2.69), UTS2 (−2.27), RNF139 (−2.13), COMMD9 (−2.09) and LOC100128356 (−2.01). The top 10 of 78 up-regulated genes in the pre-treatment group were HBB (19.5), PSME2 (13.8), CSDA (13.07), FAM127A (7.78), EXOSC1 (7.73), BPGM (7.43), CYSLTR1 (7.17), CHPT1 (6.96) and PLAC8 (6.71). qPCR analysis for CSDA, HBB, PSMiE2, PERP, PIN4, and UTS2 confirmed a similar trend to the microarray results. Interestingly absolute number of Tregs, and Th2/Treg ratio were higher in 10 IST responsive patients compared to 5 non-responsive patients (p=0.005 and 0.02, respectively). We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response. Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of Mycobacterium tuberculosis-specific Th22 cells and the effect of tuberculosis disease and HIV co-infection

2020 ◽  
Author(s):  
Mohau S. Makatsa ◽  
F. Millicent A. Omondi ◽  
Rubina Bunjun ◽  
Robert J. Wilkinson ◽  
Catherine Riou ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Latent Tuberculosis ◽  
Th22 Cells ◽  
Distinct Subset ◽  
Increased Risk ◽  
Ifn Γ

ABSTRACTThe development of a highly effective tuberculosis (TB) vaccine is likely dependent on our understanding of what constitutes a protective immune response to TB. Accumulating evidence suggests that CD4+ T cells producing IL-22, a distinct subset termed ‘Th22’ cells, may contribute to protective immunity to TB. Thus, we characterized Mycobacterium tuberculosis (Mtb)-specific Th22 (and Th1 and Th17) cells in 72 individuals with latent tuberculosis infection (LTBI) or TB disease, with and without human immunodeficiency virus (HIV)-1 infection. We investigated the functional properties (IFN-γ, IL-22 and IL-17 production), memory differentiation (CD45RA, CD27 and CCR7) and activation profile (HLA-DR) of Mtb-specific CD4+ T cells. In HIV-uninfected individuals with LTBI, we detected abundant IFN-γ producing CD4+ T cells (median: 0.93%) and IL-22-producing CD4+ T cells (median: 0.46%) in response to Mtb. The frequency of IL-17 producing CD4+ T cells was much lower, at a median of 0.06%. Consistent with previous studies, IL-22 was produced by a distinct subset of CD4+ T cells and not co-expressed with IL-17. Mtb-specific IL-22 responses were markedly reduced (median: 0.08%) in individuals with TB disease and HIV co-infection compared to IFN-γ responses. Mtb-specific Th22 cells exhibited a distinct memory and activation phenotype compared to Th1 and Th17 cells. Furthermore, Mtb-specific IL-22 was produced by conventional CD4+ T cells that required T cell receptor (TCR) engagement. In conclusion, we confirm that Th22 cells contribute substantially to the immune response to TB. Depletion of Mtb-specific Th22 cells during HIV co-infection may contribute to increased risk of TB disease.

scholarly journals Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response

2021 ◽  
Vol 22 (15) ◽  
pp. 8134
Author(s):  
Joanna Olkowska-Truchanowicz ◽  
Agata Białoszewska ◽  
Aneta Zwierzchowska ◽  
Alicja Sztokfisz-Ignasiak ◽  
Izabela Janiuk ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Cytotoxic Activity ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Peritoneal Fluid ◽  
Th17 Cells ◽  
Th2 Response ◽  
Immunoregulatory Cytokines ◽  
Ifn Γ

Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4+ T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4+ T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.

T-Bet Is Critical for the Development of Acute Graft-Versus-Host Disease Through Controlling T Cell Differentiation and Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 452-452
Author(s):  
Jianing Fu ◽  
Dapeng Wang ◽  
Yu Yu ◽  
Kane Kaosaard ◽  
Chen Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Acute Gvhd ◽  
T Cell Infiltration ◽  
Target Organs ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Abstract 452 Background: Allogeneic hematopoietic cell transplantation (HCT) offers great promise for the treatment of hematologic malignancies. However, HCT benefits are frequently offset by graft-versus-host disease (GVHD). Donor T cells can differentiate into Th1 or Th17 subset that contribute to GVHD. The T-box transcription factor T-bet is important for promoting the differentiation of naïve CD4+T cells into Th1 phenotype, while simultaneously inhibiting Th2 and Th17 lineage commitment. Published data indicate that donor T cells deficient for IFN-γ induce exacerbated GVHD. In contrast, our recent study showed that T cells deficient for T-bet were impaired in the induction of GVHD. Given T-bet is a master regulator for the differentiation into Th1 cells that produce IFN-γ, the underlining mechanisms accounted for the distinct outcomes caused by T-bet- versus IFN-γ-deficient donor T cells are not clear. Method: We evaluated the roles of T-bet and IFN-γ in acute GVHD induced by naïve CD4+ T cells or polarized Th17 cells using murine allogeneic bone marrow transplantation (allo-BMT) model. WT, T-bet knockout (KO) and IFN-γ KO mice on C57BL/6 (B6) background were used as donors, and lethally irradiated BALB/c mice were used as recipients. Pathologic analysis and serum cytokine detection were done 14 days after adoptive transfer of WT, T-bet–/–, and IFN-γ–/– CD4+ T cells. Using microarray technology, gene expression profile on donor T cells was analyzed 7 days after adoptive transfer by sorting donor-derived CD4+ T cells from the recipients of WT, T-bet–/– or IFN-γ–/– CD4+ T cells. Results: We compared the ability of WT, T-bet–/–, and IFN-γ–/– CD4 T cells in the induction of acute GVHD. In the comparison with WT cells, IFN-γ–/– CD4 T cells caused similar or even more severe GVHD as expected. In sharp contrast, T-bet–/– CD4 T cells induced much ameliorated GVHD, as significantly higher survival and less body weight loss were observed in the recipients of T-bet–/–T cells. Pathology study on GVHD target organs showed that recipients of T-bet–/– donor T cells had markedly reduced T cell infiltration and tissue damage in liver, gut, and skin, when compared with those of WT or IFN-γ–/– T cells. Reduced GVHD in the recipients of T-bet–/– T cells was consistent with significantly lower levels of pathogenic cytokines IFN-γ, TNF-α, and IL-2 but higher IL-10 (anti-inflammatory), IL-6 (Th17 related) and IL-4 (Th2 related) in serum as compared with those in the recipients of WT T cells. Mechanistic studies in vitro revealed that T-bet–/– CD4 T cells expressed significantly lower levels of IFN-γ, CXCR3 (Th1 specific chemokine receptor) and CD122 (T cell activation marker), but higher levels of IL-17 (Th17 cytokine) and CCR6 (Th17 specific chemokine receptor) compared with WT CD4 T cells, indicating that T-bet–/– T cells impaired in differentiating into Th1 cells and instead into Th17 cells. Given Th17 subset only is capable of causing GVHD and T-bet–/– T cells are prone to Th17-differentiation, we assessed the role of T-bet or IFN-γ in the development of GVHD by comparing the pathogenicity of in vitro polarized WT, T-bet–/– and IFN-γ–/– Th17 cells. While IFN-γ–/– Th17 cells had a comparable ability to cause GVHD compared with WT Th17 cells, T-bet–/– Th17 cells had reduced pathogenicity, and caused ameliorated GVHD. Furthermore, microarray analysis identified genes that are regulated by T-bet but independent of IFN-γ, including Cxcr3, Ccr5, Ccl3, Ccl4, Klrc1, Klrd1, Nkg7 and Pdcd1, which may explain the compromised ability of T-bet−/− not IFN-γ–/–T cells in the induction of GVHD. Conclusions: We conclude that T-bet is required for Th1 differentiation and optimal function of Th17 cells, and it can also control T cell infiltration into GVHD target organs by regulating chemokines and their receptors. Thus, failure in Th1 generation, migration and reduced activity of polarized Th17 cells are likely accounted for impaired ability of T-bet−/− CD4 T cells in the induction of acute GVHD. The current study suggests that targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD after allogeneic HCT in clinic. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IL-17 contributes to CD4-mediated graft-versus-host disease

Blood ◽  
2009 ◽  
Vol 113 (4) ◽  
pp. 945-952 ◽  
Author(s):  
Lucy W. Kappel ◽  
Gabrielle L. Goldberg ◽  
Christopher G. King ◽  
David Y. Suh ◽  
Odette M. Smith ◽  
...  
Keyword(s):  
T Cells ◽  
Proinflammatory Cytokines ◽  
Graft Versus Host Disease ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Interleukin 17 ◽  
Allogeneic Bone ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract CD4+ interleukin-17 (IL-17)+ T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4+ donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17−/− T-cell recipients. However, upon transfer of murine IL-17−/− CD4+ T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4+ T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17−/− CD4+ T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-γ–secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-γ, IL-4, and IL-6) in recipients of IL-17−/− CD4+ T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.

scholarly journals Characterization of Respiratory Syncytial Virus M- and M2-Specific CD4 T Cells in a Murine Model

2009 ◽  
Vol 83 (10) ◽  
pp. 4934-4941 ◽  
Author(s):  
Jie Liu ◽  
Tracy J. Ruckwardt ◽  
Man Chen ◽  
Teresa R. Johnson ◽  
Barney S. Graham
Keyword(s):  
T Cells ◽  
Respiratory Syncytial Virus ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Rsv Infection ◽  
Ifn Γ ◽  
Syncytial Virus

ABSTRACT CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2 proteins with core sequences M213-223 (FKYIKPQSQFI) and M227-37 (YFEWPPHALLV). Peptides containing the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-γ), interleukin 2 (IL-2), and other Th1- and Th2-type cytokines in an I-Ab-restricted pattern. Construction of fluorochrome-conjugated peptide-I-Ab class II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and had greater IFN-γ expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of IL-2. In addition, M209-223 peptide-activated CD4 T cells reduced IFN-γ and IL-2 production in M- and M2-specific CD8 T-cell responses to Db-M187-195 and Kd-M282-90 peptides more than M225-39 peptide-stimulated CD4 T cells. This correlated with the fact that I-Ab-M209-223 tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-Ab-M226-39 tetramer-positive CD4 T cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide-I-Ab tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.

scholarly journals Reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in graft-versus-host disease

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3101-3112 ◽  
Author(s):  
Tangsheng Yi ◽  
Ying Chen ◽  
Lin Wang ◽  
Gong Du ◽  
Daniel Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Tissue Damage ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Tissue Specific ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ifn Γ ◽  
Th17 Differentiation

In acute graft-versus-host disease (GVHD), naive donor CD4+ T cells recognize alloantigens on host antigen-presenting cells and differentiate into T helper (Th) subsets (Th1, Th2, and Th17 cells), but the role of Th subsets in GVHD pathogenesis is incompletely characterized. Here we report that, in an MHC-mismatched model of C57BL/6 donor to BALB/c recipient, WT donor CD4+ T cells predominantly differentiated into Th1 cells and preferentially mediated GVHD tissue damage in gut and liver. However, absence of interferon-γ (IFN-γ) in CD4+ T cells resulted in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin; absence of both IL-4 and IFN-γ resulted in augmented Th17 differentiation and preferential, although not exclusive, tissue damage in skin; and absence of both IFN-γ and IL-17 led to further augmentation of Th2 differentiation and idiopathic pneumonia. The tissue-specific GVHD mediated by Th1, Th2, and Th17 cells was in part associated with their tissue-specific migration mediated by differential expression of chemokine receptors. Furthermore, lack of tissue expression of the IFN-γ–inducible B7-H1 played a critical role in augmenting the Th2-mediated idiopathic pneumonia. These results indicate donor CD4+ T cells can reciprocally differentiate into Th1, Th2, and Th17 cells that mediate organ-specific GVHD.

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